Lumbar disc degeneration (LDD) is associated with both genetic and environmental factors and affects many people worldwide. A hallmark of LDD is loss of proteoglycan and water content in the nucleus pulposus of intervertebral discs. While some genetic determinants have been reported, the etiology of LDD is largely unknown. Here we report the findings from linkage and association studies on a total of 32,642 subjects consisting of 4,043 LDD cases and 28,599 control subjects. We identified carbohydrate sulfotransferase 3 (CHST3), an enzyme that catalyzes proteoglycan sulfation, as a susceptibility gene for LDD. The strongest genome-wide linkage peak encompassed CHST3 from a Southern Chinese family-based data set, while a genome-wide association was observed at rs4148941 in the gene in a meta-analysis using multiethnic population cohorts. rs4148941 lies within a potential microRNA-513a-5p (miR-513a-5p) binding site. Interaction between miR-513a-5p and mRNA transcribed from the susceptibility allele (A allele) of rs4148941 was enhanced in vitro compared with transcripts from other alleles. Additionally, expression of CHST3 mRNA was significantly reduced in the intervertebral disc cells of human subjects carrying the A allele of rs4148941. Together, our data provide new insights into the etiology of LDD, implicating an interplay between genetic risk factors and miRNA.
The nature of the cellular and molecular mechanisms for the transition of avascular cartilage replacement with bone during endochondral ossification remains poorly understood. One of the driving forces is hypoxia. As a master regulator of hypoxia, hypoxia-inducible factor-1α (HIF-1α) has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that osteoblast growth is inhibited under hypoxia and that HIF-1α cooperates with Osterix (Osx) to inhibit Wnt pathway. However, molecular mechanisms for inhibitory effects of HIF-1α on Wnt pathway are not well understood. In this study, our quantitative RT-PCR results revealed that the expression of a Wnt antagonist Sclerostin (Sost) was upregulated in osteoblasts during hypoxia while HIF-1α was upregulated. Treatment of desferrioxamine (DFO), a HIF-1α activator, led to further increase of Sost expression, suggesting that HIF-1α may activate Sost expression. The regulation of Sost gene expression by HIF-1α was then investigated. We performed loss-of-function experiments to examine Sost expression by using siRNA approach against HIF-1α, and found that the inhibition of HIF-1α by siRNA in osteoblasts led to the decrease of Sost expression. To address transcriptional regulation of Sost gene by HIF-1α, transient transfection assay was performed and showed that HIF-1α activated Sost-1 kb promoter reporter activity in a dose-dependent manner. To narrow down the minimal region of Sost promoter activated by HIF-1α, we generated a series of deletion mutants of Sost constructs. It was demonstrated that Sost-260 was the minimal region of Sost promoter for HIF-1α activation and that Sost-106 construct, which lack hypoxia response element, abolished HIF-1α-mediated Sost reporter activation. Gel shift assay showed that HIF-1 bound to the promoter sequence of Sost directly. These findings support our hypothesis that HIF-1α activates Sost expression. This study provides a novel molecular mechanism through which HIF-1α inhibits Wnt signaling in osteoblasts.
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